Article

Chemical Composition and in-Vitro Evaluation of theAntimicrobial and Antioxidant Activities of EssentialOils Extracted from Seven Eucalyptus Species

Abdul Ghaffar 1,*, Muhammad Yameen 1, Shumaila Kiran 1, Shagufta Kamal 1, Fatima Jalal 2,Bushra Munir 1, Sadaf Saleem 3, Naila Rafiq 3, Aftab Ahmad 4, Iram Saba 5 and Abdul Jabbar 5,*

Received: 27 July 2015 ; Accepted: 29 October 2015 ; Published: 18 November 2015Academic Editor: Derek J. McPhee

1 Department of Applied Chemistry and Biochemistry, Government College University, Faisalabad 38000,Pakistan; yaminsynergic@gmail.com (M.Y.); shumaila.asimch@gmail.com (S.K.);shagufta_kamal@yahoo.com (S.K.); bushramunirje@hotmail.com (B.M.)

2 Department of Zoology and Fisheries, Government College University, Faisalabad 38000, Pakistan;Fatima_jalal@hotmail.com

3 Department of Chemistry, Government College for Women University, Faisalabad 38000, Pakistan;sadafsaleemuaf@gmail.com (S.S.); n_rafiq2005@hotmail.com (N.R.)

4 Department of Biochemistry/US-Pakistan Center for Advanced Studies in Agriculture and Food Security(USPCAS/AFS), University of Agriculture, Faisalabad 38000, Pakistan; ahmadaftab1862@hotmail.com

5 Department of Chemistry, Government College University, Faisalabad 38000, Pakistan;iramsaba2005@gmail.com

* Correspondence: aghaffaruaf@yahoo.com (A.G.); a.jabbar.gpgc@gmail.com (A.J.);Tel.: +92-41-920-1032 (A.G.)

Abstract: Eucalyptus is well reputed for its use as medicinal plant around the globe. The presentstudy was planned to evaluate chemical composition, antimicrobial and antioxidant activity ofthe essential oils (EOs) extracted from seven Eucalyptus species frequently found in South EastAsia (Pakistan). EOs from Eucalyptus citriodora, Eucalyptus melanophloia, Eucalyptus crebra, Eucalyptustereticornis, Eucalyptus globulus, Eucalyptus camaldulensis and Eucalyptus microtheca were extractedfrom leaves through hydrodistillation. The chemical composition of the EOs was determinedthrough GC-MS-FID analysis. The study revealed presence of 31 compounds in E. citriodora andE. melanophloia, 27 compounds in E. crebra, 24 compounds in E. tereticornis, 10 compounds inE. globulus, 13 compounds in E. camaldulensis and 12 compounds in E. microtheca. 1,8-Cineole (56.5%),α-pinene (31.4%), citrinyl acetate (13.3%), eugenol (11.8%) and terpenene-4-ol (10.2%) were thehighest principal components in these EOs. E. citriodora exhibited the highest antimicrobial activityagainst the five microbial species tested (Staphylococcus aureus, Bacillus subtilis, Escherichia coli,Aspergillus niger and Rhizopus solani). Gram positive bacteria were found more sensitive thanGram negative bacteria to all EOs. The diphenyl-1-picrylhydazyl (DPPH) radical scavengingactivity and percentage inhibition of linoleic acid oxidation were highest in E. citriodora (82.1%and 83.8%, respectively) followed by E. camaldulensis (81.9% and 83.3%, respectively). The greatvariation in chemical composition of EOs from Eucalyptus, highlight its potential for medicinal andnutraceutical applications.

Keywords: Eucalyptus essential oils; GC/MS analysis; antimicrobial; antioxidant

1. Introduction

The interest in use of natural medicines from plants has been increasing in the past few yearsin industrialized societies, particularly against microbial agents because of the ever growing problem

Molecules 2015, 20, 20487–20498; doi:10.3390/molecules201119706 www.mdpi.com/journal/molecules

Molecules 2015, 20, 20487–20498

of antibiotic resistance [1]. The applications of essential, volatile and hydrophobic oils from naturalresources are rapidly increasing in medicinal, food and cosmetic industries to replace harmfulsynthetic additives [2,3]. The EOs are aromatic essences usually derived from the aerial parts ofthe plants containing oxygenated compounds; phenols, alcohols, esters, ethers, ketones, aldehydesand oxides, hydrocarbons; terpenes, organic sulphur and nitrogenous compounds and benzenederivatives [4,5]. EOs used as food flavorings and preservatives are generally recommended assafe (GRAS) and have broad spectrum of activity against different bacterial and fungal strains dueto the presence of high percentages of monoterpenes, eugenol, cinnamic aldehydes, thymol andpolyphenols [6,7].

Eucalyptus, with almost 900 species, is found worldwide, with some species being native toSouth East Asia. Due to its rapid growth Eucalyptus is an important hardwood forest crop used inthe pulp and paper industry. Its timber is used for construction and fuel [8], while the gum is usedfor the treatment of diarrhoea and as an astringent in dentistry [9]. More than 300 species of thisgenus contain volatile oils in their leaves [10]. Worldwide production of Eucalyptus EOs is 3000 tonsand major producers of EOs of Eucalyptus are China, Spain, Portugal, South Africa and Chile [11]).Eucalyptus EOs are used as fragrance elements in household products and cosmetics such as soaps,detergents, lotions and perfumes. They are also used as flavor elements in foods and beverages suchas baked goods, confectionaries, meat products, ice creams and soft drinks. In addition their use tohelp prevent and treat human diseases has undergone a relatively recent revival and expansion [12].

Eucalyptus EO is officially listed in the Indian Pharmacopeia as a counterirritant and mildexpectorant and also in the Chinese Pharmacopeia as a skin irritant. EOs of Eucalyptus have beenextensively used as expectorant in cough and cold compounds in various oral dosage forms,including lozenges and syrups, and as an inhalant in vapour baths. It is also used externallyfor percutaneus absorption in dosage forms including the EOs, liniments and ointments [13].EO of Eucalyptus is ingested orally to treat catarrh, used as inhalant and applied tropically as arubefacient [14].

Volatile oils of Eucalyptus are used as antioxidants and anti-inflammatory agents for skindiseases, rheumatism and as expectorant in cases of bronchitis [15,16]. Many researchers havereported the chemical composition, antioxidant and antimicrobial activities of Eucalyptus species [17].However, geographical distribution and species variation greatly affect these properties whichrequire extensive studies to explore the potential of this plant. The chemical composition of eightEucalyptus species’ essential oils from Tunisia reported the presence of 1,8-cineole as the majoringredient, followed by cryptone, α-pinene, p-cymene, α-terpineol, phellandral, cuminal, globulol,limonene, aromadendrene, sapthulenol and terpinene-4-ol [18]. 1,8-Cineole and α-pinene have beenreported as major components of EOs from seven Eucalyptus species [19].

Eucalyptus was introduced in Pakistan long ago, but interest in the tree has increasedconsiderably in the last two decades and lots of renewed efforts towards its propagation have beenmade. The large scale plantation of Eucalyptus in Pakistan has been recommended [20]. The growthof Eucalyptus in Pakistan is popular with public sector institutions, roadside plantations are widelydistributed and it is being introduced to farmers in irrigated, saline and waterlogged soil areas as apurported cash crop. Its wood has been widely used for houses and furniture. However, its leaves arenot yet used commercially. Currently Pakistan is importing more than 10 tons of Eucalyptus EOs fromdifferent countries as compared to 8.9 and 8.16 tons of Eucalyptus EOs in 1999–2000 and 2000–2001.

Presently, the volatile constituents and antioxidant activity of EOs from Pakistani Eucalyptushave not been rationally investigated and no systematic information about its chemical composition isavailable. To address this gap in our knowledge, the present study reports the chemical composition,antimicrobial and antioxidant activity of the EOs from seven Eucalyptus species for their potentialuse in food and medicine. The objectives of the study were: (i) the extraction of EOs from leavesof Eucalyptus and quantification of its yield; (ii) the identification and quantification of chemical

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compounds in each oil and (iii) determination of antioxidant and antimicrobial activities against someselected strains of bacteria and fungi.

2. Results and Discussion

2.1. Percentage Yield of EOs by Steam Distillation

The average yield of EOs from the different species of Eucalyptus was 1.84% (w/w) after 7 h ofdistillation. E. globulus had highest oil content (1.91%), while E. melanophloia contained least amount(1.73%). These findings are similar to results reporting a 2.4% yield of oil from E. gillii cultivated inIran and 2.3% in Tunisia [21,22]. The small differences may be due to different climatic, geographicand ecological parameters.

2.2. Physiochemical Analysis of Essential Oils

The physiochemical properties of seven different Eucalyptus EOs revealed that initiallyEucalyptus EOs were colourless, but after about 2 weeks they showed yellow colours (Table 1). Allthe EOs were soluble in 2 mL of 80% ethanol, but insoluble in water. The specific gravity of theEOs extracted from the seven Eucalyptus species ranged from 0.84 to 0.94. The boiling point ofE. melanophloia EO was the highest at 178 ˝C, followed by E. citriodora (177 ˝C) and the lowest was forE. globulus (161 ˝C).

Table 1. Physiochemical properties of Eucalyptus essential oils.

PhysiochemicalProperty

E.citriodora

E.camaldulensis

E.crebra

E.tereticornis

E.globules

E.melanophloia

E.microtheca

Percentage yield 1.82 1.90 1.84 1.83 1.89 1.73 1.84

Color Paleyellow

Slightlyyellowish

Lightyellow

Yellow toorange

Colorless topale yellow

Yellow tobrown

Yellowreddish,

Odor Citronellalodor

1,8 Cineoleodor

Camphorodor

Cineole-pineneodor Herbal odor Pinene odor Cymene

odor

Solubility

Insolublein water,soluble in

alcohol

Insoluble inwater, soluble

in alcohol

Insolublein water,soluble in

alcohol

Insoluble inwater,

soluble inalcohol

Insoluble inwater,

soluble inalcohol

Insoluble inwater,

soluble inalcohol

Insolublein water,soluble in

alcoholBoiling point (˝C) 177 ˝C 178 ˝C 165 ˝C 173 ˝C 161 ˝C 174 ˝C 166 ˝C

Specific gravity 0.85 0.92 0.90 0.84 0.89 0.94 0.86Refractive index 1.49 1.45 1.42 1.44 1.38 1.41 1.47

2.3. Chemical Nature of Essential Oils

The chemical composition of the seven selected Eucalyptus EOs revealed great variation in theconcentration and types of constituents (Table 2). Identification of the components of EOs was basedon their GC retention times and mass spectra, which were compared to the published data and spectraof respective standards to those constituents. The GC-MS analysis thus revealed the presence of31 components in E. citriodora and E. melanophloia, 27 components in E. crebra, 24 components inE. tereticornis, 14 components in E. globulus, 13 components in E. camaldulensis and 12 componentsin E. microtheca. The results are comparable to the 25 major compounds at an average concentrationgreater than 0.9% ˘ 0.2% reported earlier in eight Eucalyptus species EOs. E. gillii EO contained34 compounds as reported by another study [23].

Previous studies on four species of Eucalyptus grown in Tunisia reported the chemicalcompositions of E. salubris (27 compounds; 99.2%), E. salmonophloia (31 compounds; 99.2%), E. oleosa(32 compounds; 97.6%), and E. gracilis (18 compounds; 97.7%) [23].

E. citriodora and E. melanophloia EOs consisted of the highest number (31) of chemical compounds.The major components in EO of E. citriodora were citronellal (22.3%), citronellol (20%), patchoulene(9.4%), germacrene-D (7.5%), α-terpinol (6.3%), eugenol (3.9%), α-pinene (3.6%), and β-citronellal(3.2%). Previous studies on E. citriodora EO showed total 17 components out of which citronellal

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(21.2%) citronellol (12.2%) and isopulegol (11.9%) were listed as the main components [9]. Analysisof E. melanophloia EO revealed 31 components including α-pinene (16.0%), β-phellandrene (14.3%),farnesol (10.0%) and verbenol (9.2%). These results show a very efficient production of suchcompounds as compared to monoterpenoid oils, with α-pinene (1.6%), 1,8-cineole (0.2%–60%) andp-cymene (0.2%–21%) as the major constituents [24]. The chemical composition of E. camaldulensisEO showed 13 components, with major components such as linalool (17.0%), 1,8-cineole (16.1%),p-cymene (12.2%), β-farnesol (11.1%), γ-terpinene (10.7%), phenythyl acetate (7.1%), geranial (6.6%),terpinene-4-ol (5.3%), paraldehyde (5.3%) and p-ment-1-en-3, 8-diol (5.1%). EO from E. oleosa stem,immature flowers and the fruit showed 1,8-cineole as principal compound, representing 31.5%, 47.0%and 29.1%, respectively [25]. The results show small variation from a previous study describing atotal of 11 components out of which p-cymene (17.4%–28.6%), β-phellandrene (12.3%–14.4%) andβ-pinene (0.9%–11.4%) were the main ingredients [26] and great variation from the 1,8-cineole(21.7%), β-pinene (20.5%) and methyl eugenol (6.1%) levels in E. camaldulensis [27]. The studyis also comparable to a E. camaldulensis EO analysis reporting 1,8-cineole (45.7%) and p-cymene(17.1%) as major compounds obtained by hydrodistillation and 8,14-cedrane oxide (43.8%) and elemol(6.3%) by supercritical fluid extraction [28]. The findings of present study show a greater numberof oxygenated compounds. The differences may be attributed to geographical, environmental andclimatic variations affecting chemical composition of Eucalyptus species.

E. crebra oil was composed of 27 components. Limonene (14.3%) was the major constituent,followed by terpinene-4-ol (10.2%) and citrinyl acetate (9.1%). E. tereticornis EO showed24 components with 1,8-cineole (15.2%), α-pinene (12.1%), myrtenal (8.1%), linalool, (7.4%) andparaldehyde nitrile (7.1%) as the most abundant. Another study reported that E. tereticornis EOcontained 19 components out of which α-pinene (21.4%) was the major constituent but 1,8 cineole wasabsent [29]. In this study 1,8-cineole was the principal component (56.5%) found in E. globulus alongwith limonene (28.0%), α-pinene (4.2%), α-terpinol (4.0%) and globulol (2.4%). The present studyshows higher contents of these compounds as compared to earlier reports for a total of 10 componentsin this plant, out of which 1,8 cineole (17.5%), α-pinene (1.7%) and α-phellandrene (1.1%) werethe major constituents [30]. E. microtheca EO showed total 12 components, with α-pinene (31.4%),citrinyl acetate (13.2%), cymene (12.4%), eugenol (11.8%) and eucalyptol (8.1%) as the major ones. Theresults of the present study showed higher amounts than the cymene (10.3%) and α-pinene (10.0%)as the major constituents reported previously [31]. The chemical composition of EOs was statisticallysignificantly different in all Eucalyptus species. Some components were found in abundance in somespecies and absent in some species.

Table 2. Relative percentage chemical composition of essential oils of the EOs extracted from sevenEucalyptus species.

SerialNo. Components E.

citriodoraE.

camaldulensisE.

crebraE.

tereticornisE.

globulesE.

melanophloiaE.

microtheca

1. 1,8-Cineole 16.1 4.9 15.2 56.5 3.1 2.02. cis-β-Ocimene 0.1 2.1

3.4-Methylene-1-

(1-methylethyl)-3-Cyclo-hexene-1-ol

5.7

4. 3-Carene 1.75. α-Cubebene 2.2 2.2 0.26. α-Elemene 1.37. α-Humelene 2.48. α-Terpinol 6.3 49. α-Phellandrene 8.1

10. α-Pinene 3.6 0.9 2.5 12.1 4.2 16 31.411. β-Caryophyllene 1.212. β-Citronellal 3.213. β-Farnesol 11.1 2.8 1014. β-Phellandrene 0.9 14.315. β-Pinene 2.6 0.2 1.5 2.516. Amorphane 0.3

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Table 2. Cont.

SerialNo. Components E.

citriodoraE.

camaldulensisE.

crebraE.

tereticornisE.

globulesE.

melanophloiaE.

microtheca

17. Benzaldehyde 0.818. Camphene 1.1 0.1 0.919. Camphor 1.320. Citrinyl acetate 2.8 9.1 2.8 13.221. Citral 0.122. Citronellal 22.323. Citronellol 2024. Citronellal oxime 1.4 1.0 3.6 1.425. Cymene-8-ol 0.3 1.626. Cyclopentanone27. Eugenol 3.9 0.6 0.7 1.8 1.7 11.828. Eucalyptol 0.1 3.3 0.2 1.1 8.129. Digitoxigenine 1.8 1.7 0.430. Geranial 0.1 4.1 2.731. Germacrene-D 7.5 2.532. Geranial oxime 1.9 4.233. Geranial 0.1 6.6 3.6 0.1 1.434. Geranial nitrile 0.7 1.1 3.635. Geranyl acetate 2.736. Globulol 2.4 0.637. Isopulegol 0.1 2.3 0.1 0.638. Isosativene 3.7 4.539. Limonene 0.1 14.3 0.7 28 1.2 1.540. Linalool 0.5 17 1.1 7.4 0.3 0.6

41. 2-Methylprop-1-enyl-cyclohexa-1,5-diene 0.9

42. Myrtenal 0.6 8.1 9.243. Neriine 2.3 0.4 0.844. Neral 1.7 5.4 2.7 1.745. Neral oxime 1,446. Paraldehyde 5.3 6.0 1.447. Paraldehyde nitrile 1.9 5.9 7.1 0.748. Patchoulene 9.4 3.049. p-Cymene 12.2 0.2 12.450. Phenythyl acetate 7.1 0.2 1.8 1.351. Pinocarveol52. p-Ment-1-en-3,8-diol 5.153. Sabinene 4.2 1.454. Spathulenol 0.455. Solanone 0.356. Terpinene-4-ol 0.3 5.3 10.2 1.257. Verbenol 9.258. γ -Terpinene 0.7 10.759. γ-Terpinene 1 1.8 0.460. Trance-pinocarveol 1.1 6.861. γ-phellandrene 3.262. Ylangene 0.9

2.4. Antimicrobial Activity

The EOs extracted from the seven Eucalyptus species showed great potential for their applicationas antimicrobial agents. The activities were determined by using three strains of bacteria and twostrains of fungi.

2.4.1. Antibacterial Activity

The EOs extracted from all seven Eucalyptus spp. showed antibacterial activity against S. aureus,B. subtilis and E. coli (Table 3). Maximum inhibition zones were observed for the E. citriodora EOagainst Gram positive bacteria (31 mm against S. aureus and 28 mm against B. subtilis). E. melanophloiaand E. citriodora showed significant activity against the Gram negative bacterium E. coli.

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Table 3. Comparative antimicrobial activities of essential oils of Eucalyptus species.

Microbial Species Zones of Growth Inhibition ( in mm) by Eucalyptus SpeciesE. citriodora E. camaldulensis E. crebra E. tereticornis E. globules E. melanophloia E. microtheca Amoxil Nizoral

Bacterialspecies

S. aureus 31 ˘ 0.83 Aa 21 ˘ 0.851 Ade 23 ˘ 0.836 Acd 22 ˘ 0.853 Ade 28 ˘ 0.835 Acd 26 ˘ 0.836 Abc 16 ˘ 0.831 Ae 22 ˘ 0.833 Aab -B. subtilis 28 ˘ 0.833 Aa 24 ˘ 0.835 Ade 21 ˘ 0.848 Acd 18 ˘ 0.835 Ade 17 ˘ 0.833 Acd 22 ˘ 0.836 Abc 20 ˘ 0.838 Ae 28 ˘ 0.833 Aab -

E. coli 15 ˘ 0.835 Ba 10 ˘ 0.835 Bde 12 ˘ 0.835 Bcd 14 ˘ 0.836 Bde 13 ˘ 0.83 Bcd 16 ˘ 0.833 Bbc 11 ˘ 0.835 Be 20 ˘ 0.831 Bab -Fungalspecies

A. niger 29 ˘ 0.831 Aa 28 ˘ 0.835 Aab 25 ˘ 0.836 Abc 26 ˘ 0.833 Ab 24 ˘ 0.835 Abc 27 ˘ 0.835 Ac 21 ˘ 0.835 Ac - 17 ˘ 0.835 Ac

R. solani 26 ˘ 0.836 Bb 22 ˘ 0.829 Bab 19 ˘ 0.835 Bbc 21 ˘ 0.836 Bb 20 ˘ 0.835 Bbc 12 ˘ 0.835 Bc 17 ˘ 0.838 Bc - 20 ˘ 0.835 Bc

Each values is means of three. The capital letters represent significant difference in microbial species, while small letters represent significant difference in EOs.

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However, all the species showed higher activity for the Gram positive bacteria as comparedto the Gram negative bacteria. Gram positive bacteria were found to be more sensitive to E. gilliiEO and extracts than Gram negative ones [22]. E. oleosa EO exhibited an interesting antibacterialactivity against all microorganisms tested (L. monocytogenes, S. aureus, E. coli, K. pneumoniae,S. cerevisiae, C. albicans, M. ramamnianus and A. ochraceus). The activity in this study was better againstGram-positive bacteria except for S. aureus and E. coli [23]. Results have proved that E. citriodora EO ismore potent in antibacterial activity than the other six species. The EOs of Eucalyptus have previouslyshown antimicrobial and antiplasmid activities [32]. Previous studies also report that EOs againstfood spoilage organisms and food-borne pathogens are slightly more active against Gram-positivethan Gram-negative bacteria [33].

2.4.2. Antifungal Activity

E. citriodora EO was found most effective against A. niger (29 mm) and R. solani (26 mm).Significant antimicrobial activity was shown by EO of E. microtheca against A. niger (21 mm) while EOof E. melanophloia was found least effective (12 mm) against R. solani (Table 3). The results showed thatall the tested Eucalyptus EOs presented significant antifungal activity against A. niger and R. solani andhad equal or more antifungal effect than amoxil and nizoral. These properties could be correlated tothe chemical composition of the oils with good phenolic, alcoholic or aldehydic contents that may becorrelated to the geographical distribution and environmental effect on production of phytochemicalsin plants.

2.5. Antioxidant Activity

The DPPH scavenging activity was highest in E. citriodora (82.1%), followed by E. camaldulensis(81.9%) and E. microtheca (81.8%) as compared to positive control BHT. The results are comparedto the DPPH assay results for E. oleosa EO activity in the range of 12.0–52.8 mg/mL, whereas inthe 2,21-azinobis-3-ethylbenzothiazoline-6-sulfonate assay (176.5 ˘ 3.1 mg/L) it showed the bestinhibition result [18]. All the EOs strongly suppressed the peroxide formation in linoleic acid systemduring incubation. The inhibition of oxidation of linoleic acid system was higher for the EOs ofE. citriodora (83.8%) and E. camaldulensis (83.2%) than the other five Eucalyptus species (Figure 1).These findings are also supported by another study reporting 86.07% inhibition of linoleic acid fornon-polar methanol extract of E. sargentii [34].

Molecules 2015, 20, page–page

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2.4.2. Antifungal Activity

E. citriodora EO was found most effective against A. niger (29 mm) and R. solani (26 mm). Significant antimicrobial activity was shown by EO of E. microtheca against A. niger (21 mm) while EO of E. melanophloia was found least effective (12 mm) against R. solani (Table 3). The results showed that all the tested Eucalyptus EOs presented significant antifungal activity against A. niger and R. solani and had equal or more antifungal effect than amoxil and nizoral. These properties could be correlated to the chemical composition of the oils with good phenolic, alcoholic or aldehydic contents that may be correlated to the geographical distribution and environmental effect on production of phytochemicals in plants.

2.5. Antioxidant Activity

The DPPH scavenging activity was highest in E. citriodora (82.1%), followed by E. camaldulensis (81.9%) and E. microtheca (81.8%) as compared to positive control BHT. The results are compared to the DPPH assay results for E. oleosa EO activity in the range of 12.0–52.8 mg/mL, whereas in the 2,2′-azinobis-3-ethylbenzothiazoline-6-sulfonate assay (176.5 ± 3.1 mg/L) it showed the best inhibition result [18]. All the EOs strongly suppressed the peroxide formation in linoleic acid system during incubation. The inhibition of oxidation of linoleic acid system was higher for the EOs of E. citriodora (83.8%) and E. camaldulensis (83.2%) than the other five Eucalyptus species (Figure 1). These findings are also supported by another study reporting 86.07% inhibition of linoleic acid for non-polar methanol extract of E. sargentii [34].

Figure 1. Antioxidant activities of Eucalyptus EOs through linoleic acid and diphenyl-1-picrylhydazyl radical scavenging assays. (Each value is mean of three).

3. Experimental Section

3.1. Chemicals and Microbial Strains

All the chemicals used in this study were of analytical grade and purchased from Sigma Aldrich (Taufkirchen, Germany). S. aureus, B. subtilis, E. coli, A. niger and R. solani were obtained from and identified by the Department of Microbiology, University of Agriculture (Faisalabad, Pakistan). The bacterial strains were preserved and cultured separately in Lauria and Bertani (LB) media and fungal

0 20 40 60 80 100

E. citriodora

E. melanophloia

E. crebra

E. tereticornis

E. globulus

E. camaldulensis

E. microtheca

Free radical scavenging activity (Percentage inhibition of oxidation)

Euca

lypt

us sp

ecie

s

Linolic acid system(%)

DPPH scavengingactivity (%)

Figure 1. Antioxidant activities of Eucalyptus EOs through linoleic acid and diphenyl-1-picrylhydazylradical scavenging assays. (Each value is mean of three).

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3. Experimental Section

3.1. Chemicals and Microbial Strains

All the chemicals used in this study were of analytical grade and purchased from Sigma Aldrich(Taufkirchen, Germany). S. aureus, B. subtilis, E. coli, A. niger and R. solani were obtained fromand identified by the Department of Microbiology, University of Agriculture (Faisalabad, Pakistan).The bacterial strains were preserved and cultured separately in Lauria and Bertani (LB) media andfungal strains in Eggins and Pugh (E and P) medium broth by following the standard microbiologicalprotocols. All microorganisms were stocked at ´4 ˝C in standard conditions and were revived twicebefore use in the manipulations.

3.2. Collection of Samples

The fresh leaves from E. citriodora, E. melanophloia, E. crebra, E. tereticornis, E. globulus,E. camaldulensis and E. microtheca were collected from the Gutwala Forest Research Institute,(Faisalabad, Pakistan) in May 2014. All the leaves were washed with water to remove dirt particlesand shade dried to reduce the moisture contents.

3.3. Procedure to Extract EOs

The EO was extracted from each plant material through a hydrodistillation method as describedelsewhere [35]. In summary, the samples (leaves) were shade dried and a weighed amount (50 g)of crushed plant material was immediately charged to the distillation flask. Pressurized steam wascirculated through the plant material. The vapors of the pure EO along with steam were condensedwhile passing through a water condenser and collected in a receiver flask kept in ice water in orderto prevent the evaporation of the low boiling point constituents. The upper oily layer (2–3 mL) ofcondensed material was dissolved in diethyl ether (40 mL) and then separated from the distilledwater component with the help of a separating funnel. Total EO was obtained after careful removalof the solvent by evaporation. The whole process of extraction was repeated till about 14–17 mL of oilwas collected. The oils were dehydrated with Na2SO4 and the average percent yield was calculated.The EO was stored in a cool place away from heat and light.

3.4. Physiochemical Properties

The physiochemical properties of the oils like colour, odour, appearance, solubility in aqueousethyl alcohol, boiling point, specific gravity at 20 ˝C and refractive index were determined by themethods described earlier [36–39].

3.5. Chemical Composition through GC-Mass Spectroscopy

Qualitative and quantitative determination of chemical composition for each EO was carriedout through gas chromatography mass spectroscopy by using a GC-17A (Shimadzu, Kyoto, Japan)fitted with a DB-wax (30 mm ˆ 0.25 mm) column (Agilent Technologies, Waldbornn, Germany) anda flame ionization detector (FID). Injector and detector temperatures were set at 250 ˝C and 260 ˝C,respectively. Column temperature was programmed from 90 ˝C for 2 min to 180 ˝C with a gradientof 2 ˝C/min. A second gradient was applied to 240 ˝C at 3 ˝C/min. Helium was used as a carrier gasat a flow rate of 30 mL/min at 150 psi. One µL sample of each EO was injected through the injectorport. The chemical composition was determined by identifying the peaks with available data asstandard and reported as a relative percentage of the total peak area. The quantitative measurementswere made on chromatography station CSW 32 software of Data Apex (Prauge, Czech Republic,version 5.0).

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3.6. Antimicrobial Activity

The antimicrobial activity of EOs was determined by using the disk agar diffusion method. Thegrowing stock cultures were stabilized through various cycles for uniform growth. Sterilized MullerHinton agar (Sigma-Aldrich, Taufkirchen, Germany) was cooled to 50 ˝C and inoculated with 100 µLfresh culture of each one of the above mentioned bacteria (105–106 bacteria/mL), separately. Theinoculated medium (15 mL) was poured into sterilized petri dish of 9 cm diameter and swirled todistribute homogenously. Disks (9 mm diameter, Whatman filter paper no. 3) injected with 20 µLeither oil or standard antibiotics (see below) were applied on solid agar medium. The plates wereplaced at 4 ˝C for 1–2 h and then incubated at 37 ˝C for 24 h. The zones of inhibition (including thesize of the disk as well = 0.9 mm) on the media were measured with ruler [40]. Antifungal activitywas determined on Sabouraud dextrose agar medium as described earlier [41]. The microorganismswere cultured in test tubes on the agar for 24 h. Fresh Sabouraud medium was inoculated withthese spores to the desired concentration of cells (105 spores/mL) and plates were prepared. Diskscontaining 20 µL EO were applied on the medium. The plates were incubated at 30 ˝C for three daysand zones of inhibition were measured. The standard antibiotic control discs (containing 15 µg of themedicine on each disc) of Amoxil for bacteria and nizoral for fungi were used as controls, respectively.

The sensitivity of microorganism to each individual EO was determined by the diameter of thezones of inhibition with a small modification as described somewhere else [42]. Thus, the sensitivitywas characterized as follows: not sensitive for total diameters smaller than 10 mm; sensitive for totaldiameters of 10–15 mm; very sensitive for total diameters of 16–20 mm; extremely sensitive for totaldiameters larger than 20 mm.

3.7. Antioxidant Activity of EOs

3.7.1. DPPH Scavenging Activity

The free radical scavenging activities of the seven Eucalyptus EOs were assessed by measuringtheir scavenging abilities for stable 2,21-diphenyl-1-picrylhydazyl DPPH radicals [43]. To do this,the samples (0.5 µg/mL) were mixed with 1 mL of 90 µM DPPH solution and made up with 95%methanol to a final volume of 4 mL. The mixture was incubated at 25 ˝C for 1 h and absorbancewas measured at 515 nm using a spectrophotometer (U-2001, Hitachi, Tokyo, Japan). Butylatedhydroxytoluene (BHT) was used as a positive control. All the samples were analyzed in triplicate. Thefree radical scavenging activity was determined as percent inhibition by using the following equation:

Inhibition p%q “ 100´

Acontrol ´Asample{Acontrol

¯

(1)

The antioxidant activities of EOs were expressed as IC50 values, which represented theconcentrations of EOs that caused 50% neutralization of DPPH radicals and were calculated fromthe plot of inhibition percentage against concentration.

3.7.2. Antioxidant Activity in Linoleic Acid System

The antioxidant activity of EOs were also determined using inhibition of linoleic acidoxidation [44]. To do this, the test samples (50 µL) were dissolved in ethanol (1 mL) then mixedwith linoleic acid (52 µL), ethanol (4 mL) and 0.05 M sodium phosphate buffer (pH 7, 4 mL). Thesolution was incubated at 40 ˝C for 175 h. The colorimetric method was used to measure the extentof oxidation by peroxide value as described earlier [45]. For 0.2 mL sample solution, 10 mL of ethanol(75%), 0.2 mL of aqueous solution of ammonium thiocynate (30%) and 0.2 mL of ferrous chloridesolution (20 mM in 3.5% HCl) were added sequentially. The contents were stirred for 3 min and the

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absorbance was measured at 500 nm. BHT was used as a positive control. The percentage of theinhibition of linoleic acid oxidation was calculated as follows:

% inhibition of linolic acid oxidation

“ 100´„

Abs.increase of sample at 175 hAbs.increase of control at 175 h

ˆ 100(2)

3.8. Statistical Analysis

Statistical analysis was performed with the IBM Statistical Package of Social Sciences (SPSS,version 19, SPSS Inc., Chicago, IL, USA). Statistically significant differences were found by usingtwo factorial ANOVA and significant results were represented at p-values < 0.001.

4. Conclusions

Applications of natural extracts are growing rapidly in the food, cosmetic and pharmaceuticalindustry. The present study systematically explored the potential of some local Eucalyptus species.The chemical composition of EOs from selected Eucalyptus species showed great variation. Thechemical compounds of essentials oils reported in these species were also different from otherreported Eucalyptus species. The antimicrobial potential of the EOs extracted from seven Eucalyptusspecies was higher against Gram positive bacteria than Gram negative ones and two types of fungi.All the results proved that these EOs were very effective and could be used in medicines, cosmetics,food and flavors industries. This report is so far first systematic study and comparison of EOs of localprevalent Eucalyptus species and their potential for uses in health and industries.

Acknowledgments: The authors acknowledge research facilities and partial research funding provided byGovernment College University, Faisalabad, Pakistan.

Author Contributions: Abdul Ghaffar and Abdul Jabbar conceived and designed the experiments;Muhammad Yameen and Shumaila Kiran determined the physical properties of EOs; Abdul Ghaffar, Fatima Jalaland Shagufta Kamal determined chemical composition through GC-MS; Bushra Munir, Sadaf Saleem andNaila Rafiq performed antimicrobial and antioxidant activity; Aftab Ahmad prepared the manuscript andstatistically analyzed the data; Iram Saba collected the samples.

Conflicts of Interest: The authors declare no conflict of interest.

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Sample Availability: Samples of the compounds and EOs are available from the authors.

© 2015 by the authors; licensee MDPI, Basel, Switzerland. This article is an openaccess article distributed under the terms and conditions of the Creative Commons byAttribution (CC-BY) license (http://creativecommons.org/licenses/by/4.0/).

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